The Effects of Ligand Exchange and Mobility on the Peroxidase Activity of a Bacterial Cytochrome c upon Unfolding

Abstract

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c‐550 from Paracoccus versutus assessed by X‐ray crystallography and unfolding

“…This suggests enhanced flexibility in the ligand‐loop region upon replacing M100 with a Lys residue. For the wt structure the regions exhibiting elevated B‐values show good agreement with the dynamic data obtained in solution for both oxidation states of the protein [10,11].…”

“…One of these, a band at 623 nm, grows into the spectrum indicating a change from low‐ to high‐spin heme. In analogy to a previous study with the wt protein [11], we ascribe this to loss of the sixth ligand to the heme. Therefore, K100 like M100 dissociates from the heme as the protein unfolds.…”

“…The latter occur as a direct result of the dynamic equilibrium involving dissociation of the axial Met ligand. This results in deprotonated protein based ligands such as Lys, His and if available the N‐terminal α‐amino group competing for the vacant coordination site [11,31,38–40]. Owing to the paramagnetic properties of a low‐spin ferriheme, misligated heme species can be readily identified by 1 H NMR.…”

“…Regardless of which energy source is utilized, a class I c ‐type cytochrome, cyt c‐ 550, acting as an electron‐carrier is present in the respiratory chain which is homologous to the cyts c 2 found in photosynthetic bacteria [2,3]. A number of biochemical and biophysical studies on P. versutus cyt c ‐550 have been reported [4–14]. However, throughout these studies no 3D structure of the protein was available and a model based on the known structure of the cyt c‐ 550 from P. dentirificans [15] was constructed to aid in interpretation of data.…”

Protein self‐modification by heme‐generated reactive species

“…For example, the P450s are able to act as peroxygenase enzymes in the presence of alkyl peroxides through the so-called ''peroxide shunt'' (9). Even the electrontransfer protein cytochrome c, which has a six-coordinated heme and a very compact structure, when partially denaturated at acidic pH is capable of oxidizing substrates with H 2 O 2 (10,11).…”